Latex polymer particles containing fluorescent substance or contrast medium and process for producing the same

ABSTRACT

This invention provides a method to effectively incorporate inorganic fluorescent substance or inorganic contrast medium into latex polymer particles which are used for diagnostic test or the like, and also provides thus produced fluorescent substance-containing latex polymer particles which show decreased non-specific adsorption of protein or the like. Said latex polymer particles are produced by making latex-forming monomer, macromer which has at least a hydrophilic polymer segment and an inorganic fluorescent substance or an inorganic contrast medium co-existent simultaneously in an aqueous medium, and subjecting them to a polymerization reaction.

TECHNICAL FIELD

This invention relates to inorganic fluorescent substance- or inorganiccontrast medium-containing latex polymer particles which are inparticular usable for the detection of components of organisms and fordiagnosis, and to a process to produce the particles. In thisspecification, “latex polymer particles” mean polymer particles whichare capable of forming latex in aqueous medium.

BACKGROUND ART

As an example of inorganic metal-containing latex polymer particleswhich have conventionally been used for such purposes as the detectionof components of organisms, diagnosis and the like, there can bementioned particles which are manufactured by emulsion polymerizationbetween an organic layer wherein hydrophobic vinyl aromatic monomer(which may optionally include comonomer) and magnetic particles aredispersed and an emulsifier-containing aqueous solution. In suchemulsion polymerization, water-insoluble organic compounds are made toco-exist so that magnetic particles may efficiently be encapsulated inlatex particles (e.g., Patent Document 1).

“Magnetic particles (which are synthesized in the method as mentioned inPatent Document 1) have a shape of polymer-coated magnetic body, and,therefore, the magnetic particles have different sizes depending on theparticle size of magnetic body as a nucleus. Hence, it is difficult tokeep the size of magnetic particles uniform, in particular when themagnetic body has a particle size in a range of 0.1 to 1.0 μm. Besides,synthetic operation is very complicated.” For this reason, there hasbeen provided another method (e.g., Patent Document 2); latex polymerparticles which are a polystyrene- or styrene-butadiene copolymer arepreviously swollen by organic solvent and by heating, and, then,labeling material such as magnetic substance and fluorescent material isadded and mixed by stirring, and, thus, said fluorescent material or thelike is embedded in the vicinity of surface layer of the latex polymerparticles.

The method of Patent Document 1 of emulsion polymerization forencapsulating magnetic particles in latex polymer particles has somedefects. Maybe on this account, in most of other methods, swollenpolymer particles are brought into contact with an aqueous solution offluorescent substance or the like (which is to be chelated wherenecessary) so that the fluorescent substance or the like may thereby bemixed, or incorporated, into the polymer particles, and, in this manner,magnetic body or fluorescent substance is encapsulated or embedded inlatex polymer particles (e.g., Patent Documents 3 and 4). PatentDocument 3 discloses latex polymer particles which were manufacturedfrom hydrophobic monomer such as styrene, nonionic water-soluble monomersuch as acrylamide and anionic monomer such as acrylic acid, for thepurpose of improving the stability of latex polymer particles in aqueoussolution and of immobilizing physiologically reactive seed on saidpolymer particles by covalent bonding or absorption. Patent Document 4,on the other hand, uses, as a comonomer for styrene monomer or the like,a macromer which comprises poly(oxyalkylene) segment having, at oneterminal, a polymerizable ethylenic group and, at the other terminal, anactive ester group, with a view to providing a reactive microspherewhich has excellent stability in aqueous medium, and is capable ofstably immobilizing thereon functional substances such as protein bychemical bonding, and on which non-specific adsorption of protein or thelike hardly occurs.

Documents which are cited above and below are identified as follows.

Patent Document 1

-   Japanese Patent KOKAI Publication No. Sho 56 (1981)-164503 (see page    1, right lower column, lines 2 to 14, in particular)

Patent Document 2

-   Japanese Patent KOKAI Publication No. Hei 10 (1998)-55911 (see page    2, right column, lines 33 to 44, and page 5, left column, lines 34    to 45, in particular)

Patent Document 3

-   Japanese Patent KOKAI Publication No. Sho 61 (1986)-218945 (see page    4, left lower column, lines 4 to 16, and page 3, right upper column,    lines 2 to 15, in particular)

Patent Document 4

-   Japanese Patent KOKAI Publication No. Hei 8 (1996)-133990 (see page    2, left column [claim 1], the same page, right column, lines 18 to    28, in particular)

DISCLOSURE OF INVENTION

In Patent Document 2, as mentioned above, fluorescent material or thelike is embedded in the vicinity of surface layer of latex polymerparticles, simultaneously with the polymerization of bifunctionalmonomer or the like, and, thus, polymer of not large molecular weight,i.e., of the degree of oligomer, is adhered onto the surface layer ofhigh-molecular material (latex polymer particles). This suggests thatthe embedding method of Patent Document 2 alone has a possibility thatthe embedded fluorescent material or the like may be released frompolymer particles by washing or the like. Patent Document 4 mentions anidea of impregnating the core portion of reactive microsphere with dyeor pigment for use as functional dye or the like. Patent Document 4,however, neither has any concrete mention of how to impregnate nor givesany description of reactive microsphere whose core portion was actuallyimpregnated with dye or pigment. Patent Document 4 refers to microsphereon which non-specific adsorption of protein or the like hardly occurs.If, however, further improvement is possible, it would be desirable toprovide means to that end.

Thus, the first objective of this invention is to provide a method toefficiently and stably include fluorescent substance or contrast mediumin latex polymer particles (in particular such ones as mentioned inPatent Document 4, which have, on the surface layer of particles, adomain originated in macromer which gives hydrophilicity to polymerparticles). Another objective of this invention is to provide latexpolymer particles which stably contain fluorescent substance or contrastmedium, and which show much decreased non-specific adsorption ofundesirable protein or the like thereon.

The inventors of this invention have assiduously studied how to attainthe above-mentioned objectives. As a result, they have found thatinorganic fluorescent substance or inorganic contrast medium isefficiently and stably encapsulated or taken into polymer particles whensuch fluorescent substance or contrast medium is made to co-exist duringthe formation of latex polymer particles by the copolymerization of alatex-forming monomer and a macromer having water-soluble (orhydrophilic) polymer segment, in contrast to Patent Document 1 whereinnot only magnetic particles but also water-insoluble organic compoundsare made to co-exist during emulsion polymerization. The inventors havefurther found out that, when there are used, for the above-mentionedmacromer, at least two kinds of macromers each having apoly(ethyleneglycol) segment which has at its one terminal a specificfunctional group, non-specific adsorption of undesirable protein or thelike can significantly be decreased as compared with the case where asingle kind macromer is used.

Thus, this invention provides a method to produce fluorescentsubstance-containing latex polymer particles, characterized in thatpolymerization reaction is conducted in an aqueous medium while theaqueous medium is stirred, said aqueous medium comprising:

-   (i) one or more kinds of latex-forming monomers,-   (ii) a macromer which has, on one terminal, a polymerizable    ethylenic group and has, on the other terminal, a hydrophilic    polymer segment which is linked or not linked by a hydrophobic    polymer segment,-   (iii) a radical polymerization initiator, and-   (iv) an inorganic fluorescent substance or an inorganic contrast    medium.

This invention also provides, as another embodiment, hydrophobiccore-hydrophilic shell type latex polymer particles which include, intheir hydrophobic core domain, inorganic fluorescent substance orinorganic contrast medium, said latex polymer particles having anaverage particle size of 0.001 to 5 μm, and being formed by radicalpolymerization in an aqueous medium which comprises:

-   (a) 0.5 to 99.5% by weight of one or more kinds of latex-forming    monomers,-   (b) 0.5 to 99.5% by weight of macromer which has, on one terminal, a    polymerizable ethylenic group and has, on the other terminal, a    hydrophilic polymer segment which is not linked by a hydrophobic    polymer segment [this macromer includes at least two kinds of    macromers each of which has, on said the other terminal, a    poly(ethyleneglycol) segment which carries a group selected from the    group consisting of hydroxyl group, carboxyl group, aldehyde group,    amino group, imino group, mercapto group, active ester-type    protected hydroxyl group, active ester-type protected carboxyl    group, acetal-type protected aldehyde group, organic    sulfonyl-protected hydroxyl group, reactivity-protected amino group    and C₁-C₄ alkoxyl group, recurring unit of said ethyleneglycol being    5 to 1200].

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is ¹H-NMR spectrum of macromer which was obtained in MacromerSynthesis Example 1.

FIG. 2 is ¹H-NMR spectrum of macromer which was obtained in MacromerSynthesis Example 2.

DESCRIPTION OF EMBODIMENTS OF INVENTION

In this specification, “latex polymer particles” mean polymer particleswhich are capable of forming latex in aqueous medium. The term latex isused in a sense which is common among those skilled in the art. Itmeans, for instance, a dispersion of polymer particles in water as adispersing medium. The term aqueous medium means an aqueous solutionwhich may contain water-miscible organic solvent, e.g., ethanol,methanol, tetrahydrofuran, acetone and acetonitrile, and also bufferetc. In a specific example, the aqueous medium is pure water.

“Inorganic fluorescent substance” is used in a sense which isinterchangeable with fluorescent material or fluorogenic material, i.e.,a material which emits remarkable luminescence with various stimulationfrom outside. Contrast medium means nuclear magnetic resonance imaging(MRI) agent or X-ray contrast medium. Non-restrictive, examples ofinorganic fluorescent substance and inorganic contrast medium includerare-earth metals which belong to lanthanoid in periodic table of theelement of the element, e.g., europium (Eu), terbium (Tb), samarium (Sm)and gadolinium (Gd), and, furthermore, certain kinds of semiconductorsuch as CdS, CdSe and InP. Rare-earth metals may be contained, in theform of chelate compound or chelate complex, in latex polymer particlesof this invention. Fluorescent substance-containing latex particles meanpolymer particles produced by copolymerization using latex-formingmonomer having polymerizable ethylenic group and macromer as mentionedlater, the hydrophobic core domain of which has encapsulated or taken inall or part of fluorescent substance, and from which the fluorescentsubstance is hardly or not released at all by usual washing or the like.

Examples of chelating agent for rare-earth metals which isadvantageously usable for the formation of such particles include1,3-diketones such as thenoyltrifluoroacetone, benzoylacetone andacetylacetone, which are not restrictive. In particular, when Gd is usedas contrast medium, complexes which are sold on the market as finalproducts under general name such as meglumine gadopentetate andgadodiamide hydrate are usable as they are. Furthermore, when rare-earthmetal is to be used for fluorescent emission, Lewis base such astrioctylphosphine oxide (TOPO) and phenanthroline (Phen) may be usedwith rare-earth metal compound. Besides, barium, barium salt, etc.,which are not rare-earth metal, are included in the above-mentionedcontrast medium.

In this invention, “latex-forming monomer” includes any known monomerthat is capable of forming latex by radical polymerization in aqueousmedium, so long as it serves to achieve the objective of this invention.“Known” monomer means that it is publicly known in this field byliteratures such as Patent Documents 1, 2 and 3 as mentioned above. Notrestrictive, examples of such a monomer include hydrophobic vinylmonomer, in particular vinyl aromatic compounds such as substituted orunsubstituted styrene and 1-vinylnaphthalene, more specifically styrene,α-methylstyrene, ethylstyrene, p-bromostyrene, vinyltoluene andt-butylstyrene. Also included are C₁-C₄ alkyl (meth)acrylate, morespecifically methyl acrylate, methyl methacrylate, ethyl acrylate, ethylmethacrylate, n-butyl acrylate and n-butyl methacrylate. Furthermore,substituted or unsubstituted conjugated diene, e.g., butadiene etc., isalso included. Such a hydrophobic monomer is preferably used in anamount of 0.5 to 99.5% by weight, desirably 10 to 90% by weight, moredesirably 20 to 80% by weight, based on the total polymer weight oflatex polymer particles of this invention. Among the above-listedmonomers, substituted or unsubstituted styrene is generally mostsuitable. When polymerizable ethylenic group of macromer which ismentioned later is aromatic monomer-originated group, also C₁-C₄ alkyl(meth)acrylate is desirably used. Examples of C₁-C₄ alkyl includemethyl, ethyl, n-propyl, isopropyl, n-butyl and t-butyl. These monomersmay be used in combination of two or more monomers. Incidentally, aswould clearly be seen from the above-mentioned examples of (meth)acrylicesters, “(meth)acrylic acid” means acrylic acid, methacrylic acid orboth.

The above-mentioned hydrophobic monomer is essential as latex-formingmonomers in this invention. Said monomers may contain, as optionalcomponent, water-soluble monomer. Representative examples ofwater-soluble monomer are amide (meth)acrylate, 2-hydroxyethyl(meth)acrylate, 2-hydroxypropyl (meth)acrylate and N-vinylpyrrolidone,which may substitute for a part, e.g., 0 to 99%, of the above-mentionedamount of hydrophobic monomer. Furthermore, crosslinking monomer, forexample a monomer corresponding to divinyl compound of theabove-mentioned vinyl aromatic compound, or to biscompound of(meth)acrylic ester, specifically divinyl benzene,bis(meth)acryloylethyl, etc., may, as optional component, substitute fora part, e.g., 0 to 99%, of the above-mentioned amount of hydrophobicmonomer.

This invention uses, as another essential monomer which constitutespolymer of latex polymer particles, a macromer which has at leasthydrophilic polymer segment. Macromer, which is also calledmacromonomer, usually means a polymerizable polymer whose molecularweight is thousands to tens of thousands. In this invention, macromeralso includes so-called oligomer whose molecular weight is hundreds. Themacromer which is used in this invention has hydrophilic polymer (whichincludes oligomer; the same applies in the following) segment as anessential constituent segment. Hydrophilic segment means a segmentcomprising a polymer chain which becomes water-soluble as acorresponding independent polymer, not as a segment in macromer.

Such a hydrophilic segment is preferably nonionic, and, although notrestrictive, comprises a main chain such as poly(ethyleneglycol)[hereinafter sometimes referred to as PEG; interchangeable withpoly(oxyethylene) or poly(ethyleneoxide)], poly(vinylalcohol),poly(vinylpyrrolidone), poly(dextran), poly(dextrin), gelatin, etc. Inthe above-mentioned macromer, such a hydrophilic segment is connected,at one terminal, to a polymerizable ethylenic group via a suitablelinking group. “Polymerizable ethylenic group” means a group which iscapable of proceeding with reaction under normal radical polymerizationreaction condition. Hence, not restrictive, polymerizable ethylenicgroup may be a group which can be present in a residue originated inmonomers as listed above with respect to latex-forming monomer. Examplesof such residue include (meth)acryloyl and vinylbenzyl, vinylphenyl,etc., whose aromatic ring is substituted or unsubstituted. Theabove-mentioned hydrophilic polymer segment, although dependent on howto prepare a corresponding macromer, is linked to the above-mentionedresidue via a linking group which comprises at least one of oxygen orsulfur atom, carbonyl, carbonyloxy, oxycarbonyl, imino, carbonylimino,iminocarbonyl, C₁-C₄ alkylene and C₁-C₄ alkynylene, to make macromer ora part of macromer which is used in this invention. Such a macromer or apart thereof can be formed by known selective termination ofcorresponding water-soluble polymer with the above-mentioned residue;for example, in the case of synthetic polymer segment, by the terminaltreatment of living polymer with (meth)acrylic acid or its reactiveester, or by living polymerization by using, as a living polymerizationinitiator, alcohols such as vinylbenzylalcohol; or in the case ofnatural water-soluble polymer segment, on the other hand, by selectivetreatment of one terminal of a corresponding polymer with use of(meth)acrylic acid or its reactive ester. There can be employed any ofthese treatments or reactions that are well known to skilled persons.

In the macromer which is used in this invention, a hydrophilic polymersegment may be linked by hydrophobic polymer segment as well as theabove-mentioned linking group. In this specification, hydrophobicpolymer segment means a concept opposite to the aforementionedhydrophilic polymer segment. Concretely, it is understood that anindependent polymer corresponding to hydrophobic polymer segment isscarcely-soluble or insoluble in water. Not restrictive, examples ofsuch a hydrophobic polymer segment include poly(lactide) chain,poly(ε-caprolactone) chain, poly(α- and/or β-benzyl aspartic acid) chainand poly(γ-benzyl glutamic acid) chain. Examples of polymer or macromerwhich has poly(ethyleneglycol) chain as a hydrophilic polymer segment,and has a chain listed above as a hydrophobic polymer segment arementioned in U.S. Pat. No. 5,449,513 (Japanese Patent KOKAI PublicationNo. Hei 6-107565) and U.S. Pat. No. 5,925,720 (WO 96/33233). Thesesegments may have a length which ranges from that of the above-mentionedoligomer to that of polymer, so long as the objective of this inventionis achieved. In the case of PEG for instance, ethyleneglycol unit ispreferably within a range of 5 to 1200. Anyone skilled in the art wouldbe able to decide the chain length of any hydrophilic polymer segmentother than PEG, in the light of the above-explained chain length of PEG.Hydrophobic polymer segment, on the other hand, can have a chain lengthof 0 to 500, or, when the polymer segment exists, 5 to 500. When such ahydrophobic polymer segment exists, it may be linked to a residue asmentioned above such as vinylbenzyl whose aromatic ring is substituted(by, for instance, C₁-C₄ alkylene, halogen atom, etc.) or unsubstitutedand (meth)acryloyl via a linking group which comprises at least one ofoxygen or sulfur atom, carbonyl, carbonyloxy, oxycarbonyl, imino,carbonylimino, iminocarbonyl, C₁-C₄ alkylene and C₁-C₄ alkynylene.Hydrophobic polymer segment and hydrophilic polymer segment may belinked to each other either directly or via the above-mentioned linkinggroup.

An especially preferably macromer which is usable in this invention hasno hydrophobic polymer segment, and has PEG as a hydrophilic polymersegment. This hydrophilic polymer segment serves to decrease theabove-mentioned non-specific adsorption of protein or the like.Conveniently usable PEG segment carries, at an other terminal than theone which has a polymerizable ethylenic group, a reactive functionalgroup such as hydroxyl group, aldehyde group, carboxyl group, aminogroup, imino group, mercapto group, active ester-type protected hydroxylgroup, active ester-type protected carboxyl group, acetal-type protectedaldehyde group and reactivity-protected amino group (e.g., maleimide) orthe like, which, under circumstances, may form, after the deblocking ofprotective group, a covalent bond with a functional group which existsin biomolecule like protein, nucleic acid, sugars and composite thereof.Examples of the group at said other terminal of PEG also include C₁-C₄alkoxyl group so as to inhibit reactivity or interaction withbiomolecule, or organic sulfonyl-protected hydroxyl group which canchemically be converted into another functional group. Although notrestrictive, examples of organic sulfonyl include tosyl and mesyl. Theterm “active ester-type protected” group means, as is well known toskilled persons, that said protected hydroxyl and carboxyl groups areeach so protected as to easily form an ester with carboxyl group andamino or hydroxyl group in the above-mentioned biomolecule,respectively. An example of macromer which has an active ester-typeprotected hydroxyl group is given in the above-mentioned Patent Document4. The above-mentioned WO 96/33233 refers to a macromer havinghydrophobic polymer segment, and to a method to introduce multi-reactivefunctional group at a terminal. Thus, in accordance with thesedocuments, any skilled person would be able to manufacture various kindof macromers which have no hydrophobic polymer segment as stated above.Representative examples of such macromer (which may contain hydrophobicpolymer segment) have formula (I) as follows:

wherein R denotes hydrogen atom or C₁-C₄ alkyl group; L₁ denotes alinking group comprising a portion other than vinyl group of radicallypolymerizable monomer, e.g., methylene, substituted or unsubstitutedphenylene or phenyl alkylene, oxy, carbonyl, carbonyloxy, and acombination thereof; B denotes a moiety of the following structure:

L₂ denotes a linking group comprising oxygen atom, C₁-C₄ alkylene,carbonyl, imino, or a combination of at least two thereof;X denotes hydrogen atom, C₁-C₄ alkyl, C₁-C₄ alkylenecarboxyl, C₁-C₄alkylenecarboxyl ester (said ester is exemplified by acid halide, C₁-C₄alkyl ester and other active ester), C₁-C₄ alkyleneamino, C₁-C₄alkylenemercapto, C₁-C₄ alkyleneacetal (e.g.:

wherein R¹ denotes hydrogen atom or C₁-C₄ alkyl) or C₁-C₄alkyleneoxycarbonylimidazol (e.g.:

wherein R² denotes hydrogen atom or C₁-C₄ alkyl); m denotes an integerof 0 to 500, and n denotes an integer of 5 to 1200.

Especially preferable among the above-mentioned macromers is acombination of a macromer (hereinafter also referred to as non-reactivePEG macromer) wherein m denotes 0 (i.e., no hydrophobic segment iscontained), and wherein X denotes hydrogen atom or C₁-C₄ alkyl (saidalkyl is exemplified by such ones as have been explained with regard to(meth)acrylic ester), and at least one macromer (hereinafter alsoreferred to as reactive PEG macromer) wherein X denotes neither hydrogenatom nor C₁-C₄ alkyl. Fluorescent substance-containing latex polymerparticles which are produced from a combination of two kinds ofmacromers as mentioned above are not described in any prior artreferences, and are therefore novel so far as the inventors know. Suchpolymer particles which are produced from two kinds of macromers asmentioned above show unexpected effects of significant decreasing of theundesirable non-specific adsorption of protein onto the surface ofparticles, as compared with, for instance, functionalmaterial-immobilized microsphere as disclosed in Patent Document 4 whoserecurring units comprise, as macromer, reactive PEG macromer alone. Suchnovel fluorescent substance- or contrast medium-containing latex polymerparticles are conveniently manufactured by the method of this inventionas mentioned above. In order to introduce fluorescent substance orcontrast medium into core portion, one may employ another methodaccording to which any known latex polymer particles that containneither fluorescent substance nor contrast medium are previouslyprepared, and, later, fluorescent substance or contrast medium isintroduced by any suitable method.

Thus, this invention provides, as another embodiment, hydrophobiccore-hydrophilic shell type latex polymer particles which containfluorescent substance in hydrophobic core domain, said latex polymerparticles not limited to the production process of this invention.Incidentally, non-reactive PEG macromer and reactive PEG macromer arepreferably combined so that the PEG chain length of the non-reactive PEGmay be the same as, or shorter than, that of the reactive PEG. The PEGchain length of the non-reactive PEG is usually 20 to 100%, preferably40 to 90%, of that of the reactive PEG. The molar proportion ofnon-reactive PEG macromer to reactive PEG macromer ranges from 1:5000 to5000:1, preferably from 1:3000 to 3000:1, especially desirably from1:100 to 1000:1. Latex polymer particles (which may contain fluorescentsubstance) having recurring units originated in two kinds of PEGmacromers in the above-mentioned proportion remarkably decreasenon-specific adsorption of protein or the like to the surface of theparticles. Such particles are therefore especially preferably handled invivo or in vitro with organism-originated sample. Furthermore, polymerparticles prepared from two kinds of macromers improve the bonding ofreactive PEG macromer-originated functional group to biomolecule. Saidone or more kinds of macromers are preferably used in an amount of 0.5to 99.5% by weight, desirably 10 to 90% by weight, more desirably 20 to80% by weight, based on the total weight of latex polymer particles(which contain no fluorescent substance).

According to the method of this invention, the above-mentionedlatex-forming monomer and the above-mentioned macromer are subjected topublicly known radical polymerization in aqueous medium. During saidradical polymerization, fluorescent substance (as a chelated compoundunder circumstances) is made to co-exist in an amount of 0.001 to 90% byweight, preferably 0.1 to 60% by weight, especially desirably 1 to 20%by weight, based on the total weight of the above-mentioned monomer andmacromer.

Radical polymerization reaction is conducted among the above-mentionedlatex-forming monomer, macromer, fluorescent substance and radicalpolymerization initiator put in an aqueous medium, optionally withheating (up to about 100° C.). This reaction system is usually placed ininert atmosphere such as argon, nitrogen or the like. Said latex-formingmonomer in an aqueous medium is conveniently chosen so that it mayaccount for 0.1 to 50 w/w %. The above-mentioned reaction system isprepared in any order so long as polymerization reaction proceeds.Preferably, however, it should follow Examples which are mentionedlater. Optimal conditions of reaction time vary depending on reactiontemperature and the species of monomer. Generally, however, reaction iscarried out for 24 hours. For radical polymerization initiator, anyconventional initiator will do, without restriction. Representativeexamples of radical polymerization initiator include azo compounds suchas

-   2,2′-azobisisobutyronitrile (AIBN),-   2,2′-azobis[2-(2-imidazolin-2-yl)propane],-   2,2′-azobis(2-methylbutyronitrile); and organic peroxides such as    benzoyl peroxide, t-butyl hydroperoxide and di-isopropyl    peroxydicarbonate. Such initiator may account for 0.001 to 10 mole    %, preferably 1 to 5 mole %, based on the total monomer (including    macromonomer).

Thus produced latex polymer particles may be purified by centrifugalseparation, sedimentation, dialysis, ultrafiltration or gel filtration,either separately or in combination. Among thus obtained latex polymerparticles, those which have reactive PEG macromer-originated unit canhave antibody, antigen, haptene, lectin or sugar immobilized thereon viacovalent bond through any known reaction, after protective group (e.g.,acetal) is deblocked where necessary. Hence, especially when two kindsof macromers are used in vivo as a targeting label or in vitro,non-specific adsorption of protein or the like hardly or not occurs atall, and, therefore, the particles are usable as an assaying system withlow background.

In the following, this invention is specifically explained by workingexamples, which do not restrict the scope of this invention.

<Measurement Apparatus and Conditions etc.>

(1) Measurement of molecular weight:

Gel permeation chromatography (GPC) HLC-8020 made by Tosoh Corporation,Detector: Refractive Index Detector RID-6A, Column: TSK-gel (superHZ-2500, super HZ-3000, super HZ-4000), Mobile phase: 2%triethylamine-containing THF, Flow rate: 1 mL/min.

(2) Nuclear Magnetic Resonance Spectrum (¹H-NMR)

JEOL EX-400 (400 MHz) made by Japan Electron Optics Laboratory Co.,Ltd., Solvent: DMSO-d₆, Measurement temperature: 20° C.

(3) Measurement of particle size:

Dynamic Light Scattering (DLS) Photometer (DLS-7000) made by OtsukaElectronics Co., Ltd.

Light Source: Ar laser

(4) Measurement of intensity of fluorescence:

Spectrofluorophtometer F-2500 made by Hitachi, Ltd. Each of the obtainedparticle suspension was diluted to 1/500 with super high-purity water,and was subjected to measurement of intensity of fluorescence under thefollowing measurement conditions, and, thus, intensity of fluorescenceper gram of particle was calculated (based on Comparative Example 1below as a standard). Photomultiplier voltage: 700 V, Excitation wavelength: 340 nm, Fluorescence wave length: wave length which showsmaximum intensity (615 to 616.5 nm)

MACROMER SYNTHESIS EXAMPLE 1 Synthesis of VB-PEG-NH₂

Process to Prepare Acetone-Potassium:

A reactor was fed with 35.2 mL of tetrahydrofuran (THF), 5 mL (15 mmol)of 3M potassium hydride (KH)/THF solution, 0.735 mL (10 mmol) of acetonein argon atmosphere at room temperature. The resultant mixture wasstirred for 15 minutes to give 0.25 M acetone-potassium/THF solution.

Process to Synthesize VB-PEG-NH₂

A reactor was fed with 1 mL (2 mmol) of 2M vinylbenzylalcohol (VBA)/THFsolution and 8 mL (2 mmol) of 0.25 M acetone-potassium/THF solution inargon atmosphere at room temperature. The resultant mixture was stirredfor 15 minutes to give a solution of potassium alkoxide of VBA. Fromthis reaction mixture, acetone was evaporated by vacuum drying. Later,60 mL of THF was added, and, moreover, 11.3 mL (0.23 mol) ofethyleneoxide was added by cooled syringe. The resultant mixture wasstirred for two days at room temperature to cause ring-openingpolymerization, and, thus, VB-PEG-OH was synthesized.

To this ring-opening polymerization reaction product, 1.3 mL (9.4 mmol)of triethylamine was added. The resultant solution is hereinafterreferred to as solution A. There was added 0.5 mL (6.5 mmol) ofmethanesulfonyl chloride to 10 mL of THF. The resultant solution ishereinafter referred to as solution B. Solution A was added dropwise tosolution B over a period of about one hour. After the dropwise additionwas over, the resultant mixture was stirred for further two hours. Then,this reaction mixture solution was filtrated. Filtrate was poured toether to precipitate monomer. The macromer was separated by filtration,and solvent was evaporated by vacuum drying, and, thus,VB-PEG-methanesulfonyl (VB-PEG-Ms) was obtained.

In 110 mL of distilled water, 9.0 g of VB-PEG-Ms (2.34 mmol) wasdissolved. The resultant solution is hereinafter referred to as solutionC. Solution C was added dropwise to 500 mL of 25% ammonia water over aperiod of about one hour at room temperature. After the dropwiseaddition was over, the resultant mixture was stirred for three days atroom temperature. From this reaction solution, ammonia was evaporated byevaporator, and, furthermore, the solution was concentrated to about 100mL. This concentrated solution was poured to isopropyl alcohol which hadbeen cooled to −15° C., and, thus, monomer was precipitated. Macromerwas recovered by centrifugal separation (6000 r.p.m, 40 minutes, −10°C.). Thus obtained macromer was dissolved in benzene. After theresultant solution was freeze-dried, macromer (Compound 1, also referredto as VB-PEG-NH₂) was recovered.

The obtained compound was confirmed by gel permeation chromatography(GPC) (with HLC-8020 made by Tosoh Corporation) and nuclear magneticresonance measurement apparatus (JEOL EX-400 (400 MHz) made by JapanElectron Optics Laboratory Co., Ltd.) under the above-mentionedmeasurement condition. From the result of GPC, it was known that PEGchain had a molecular weight of 3590, and a molecular weightdistribution Mw/Mn of 1.04.

FIG. 1 shows ¹H-NMR spectrum of VB-PEG-NH₂. From the ¹H-NMR spectrum,vinyl group-introducing rate and amino group-introducing rate werecalculated, and, thus, it was confirmed that vinyl group and amino grouphad been almost quantitatively introduced.

MACROMER SYNTHESIS EXAMPLE 2 Synthesis 2 of VB-PEG-NH₂

Macromer (corresponding to Compound 1) was synthesized with use ofpotassium hydride (KH)/THF solution in place of acetone-potassium THFsolution of the above Synthesis Example 1.

Preparation of KH/THF Solution:

A vessel was fed with KH/oil in an argon atmosphere, and, then, oilcontent was removed with hexane. This operation was repeated threetimes, and, then, vacuum drying was conducted overnight to completelyremove hexane. THF was added, and, thus, 3M KH/THF solution wasprepared.

Process to Synthesize VB-PEG-NH₂

A reactor was fed with 58 mL of THF, 1 mL (2 mmol) of 2M VBA/THFsolution and 0.8 mL (2.4 mmol) of 3 M KH/THF solution in argonatmosphere at room temperature. The resultant mixture was stirred for 30minutes at room temperature to give a solution of potassium alkoxide ofVBA. This solution was left to stand still for two hours to precipitateexcessive KH, and, then, supernatant solution was put in a vessel inargon atmosphere. Then, 11.3 mL (0.23 mol) of ethyleneoxide was added byusing cooled syringe. The resultant mixture was stirred for two days atroom temperature to cause ring-opening polymerization, and, thus,VB-PEG-OH was synthesized.

To this ring-opening polymerization reaction product, 1.3 mL (9.4 mmol)of triethylamine was added. The resultant solution is hereinafterreferred to as solution A. There was added 0.5 mL (6.5 mmol) ofmethanesulfonyl chloride to 10 mL of THF. The resultant solution ishereinafter referred to as solution B. Solution A was added dropwise tosolution B over a period of about one hour. After the dropwise additionwas over, the resultant mixture was stirred for further two hours. Then,this reaction mixture solution was filtrated. Filtrate was poured toether to precipitate macromer. The macromer was separated by filtration,and solvent was evaporated by vacuum drying, and, thus, VB-PEG-Ms wasobtained.

In 110 mL of distilled water, 7.7 g of VB-PEG-Ms (1.4 mmol) wasdissolved. The resultant solution is hereinafter referred to as solutionC. Solution C was added dropwise to 500 mL of 25% ammonia water over aperiod of about one hour at room temperature. After the dropwiseaddition was over, the resultant mixture was stirred for three days atroom temperature. From this reaction solution, ammonia was evaporated byevaporator, and, then, the solution was concentrated to about 100 mL.This concentrated solution was poured to isopropyl alcohol which hadbeen cooled to −15° C., and, thus, macromer was precipitated. Monomerwas recovered by centrifugal separation (6000 r.p.m, 40 minutes, −10°C.). Thus obtained macromer was later dissolved in benzene. After theresultant solution was freeze-dried, macromer was recovered as desired.

The obtained compound was confirmed by gel permeation chromatography(GPC) (with HLC-8020 made by Tosoh Corporation) and nuclear magneticresonance measurement apparatus (JEOL EX-400 (400 MHz) made by JapanElectron Optics Laboratory Co., Ltd.) under the above-mentionedmeasurement condition. From the result of GPC, it was known that PEGchain had a molecular weight of 5460, and a molecular weightdistribution Mw/Mn of 1.03.

From the ¹H-NMR spectrum, vinyl group-introducing rate and aminogroup-introducing rate were calculated, and, thus, it was confirmed thatvinyl group and amino group had been almost quantitatively introduced.

MACROMER SYNTHESIS EXAMPLE 3 Synthesis of VB-PEG-OH

A reactor was fed with 1 mL (2 mmol) of 2M VBA/THF solution and 8 mL (2mmol) of 0.25 M acetone-potassium/THF solution in argon atmosphere atroom temperature. The resultant mixture was stirred for 15 minutes togive a solution of potassium alkoxide of VBA. From this reactionmixture, acetone was evaporated by vacuum drying. Later, 60 mL of THFwas added, and, moreover, 6.8 mL (0.14 mol) of ethyleneoxide was addedby using cooled syringe. The resultant mixture was stirred for two daysat room temperature to cause ring-opening polymerization. Then, 3 mL ofmethanol was added to stop reaction. This reaction mixture solution waspoured to isopropyl alcohol which had been cooled to −15° C., and, thus,macromer was precipitated. Macromer (Compound 2) was recovered bycentrifugal separation (6000 r.p.m, 40 minutes, −10° C.). Solvent wasremoved by freeze drying.

The obtained compound (hereinafter referred to as Compound 2 orVB-PEG-OH) was confirmed by gel permeation chromatography (GPC) (withHLC-8020 made by Tosoh Corporation) and nuclear magnetic resonancemeasurement apparatus (JEOL EX-400 (400 MHz) made by Japan ElectronOptics Laboratory Co., Ltd.) under the above-mentioned measurementcondition. FIG. 2 shows ¹H-NMR spectrum of VB-PEG-OH. From the result ofGPC, it was known that PEG chain had a molecular weight of 2850, and amolecular weight distribution Mw/Mn of 1.04.

From the ¹H-NMR spectrum, vinyl group-introducing rate was calculated,and, thus, it was confirmed that vinyl group had been almostquantitatively introduced.

MACROMER SYNTHESIS EXAMPLE 4 Synthesis of Acetal-PEG/PLA-Methacryloyl

Process to Prepare Potassium-Naphthalene/THF Solution:

To a reactor which contained naphthalene in argon atmosphere, THF wasadded and dissolved. Then, under ice cooling, pillar-shaped potassiumwas added in a molar amount of 1.05 times as much as naphthalene, andthe resultant mixture was stirred for one day. The resultant solutionwas titrated with hydrochloric acid to give 0.3263 Mpotassium-naphthalene/THF solution.

Synthesis of Acetal-PEG/PLA-Methacryloyl

A reactor was fed with 40 mL of THF and 0.32 mL (2 mmol) of3,3′-diethoxy-1-propanol in argon atmosphere at room temperature. Then,6.2 mL (2 mmol) of 0.3263 M potassium-naphthalene/THF solution wasadded, and the resultant mixture was stirred for 15 minutes to give asolution of potassium alkoxide solution. To this solution, 11.3 mL (0.23mmol) of ethyleneoxide was added by cooled syringe. The resultantmixture was stirred for two days at room temperature to causering-opening polymerization, and, thus, acetal-PEG-OH was synthesized.To this polymerization solution, 8.4 mL (8.4 mmol) of 1 mol/LDL-lactide/THF solution was added, and stirred for three hours at roomtemperature for further polymerization reaction to occur. Subsequently,4.5 mL (28 mmol) of methacrylic anhydride was added, and, after stirringfor two days at room temperature, reaction was stopped. This macromermixture solution was poured to isopropyl alcohol which had been cooledto −15° C., and, thus, macromer was precipitated. Macromer was recoveredby centrifugal separation (6000 r.p.m, 40 minutes, −10° C.). Macromerwas further poured to isopropyl alcohol to precipitate. Then, macromerwas purified by centrifugal separation (6000 r.p.m, 40 minutes, −10°C.), and, subsequently, macromer was dissolved in benzene. Byfreeze-drying, macromer (Compound 3, also referred to asacetal-PEG/PLA-methacryloyl) was recovered.

The obtained compound was confirmed by gel permeation chromatography(GPC) (with HLC-8020 made by Tosoh Corporation) and nuclear magneticresonance measurement apparatus (JEOL EX-400 (400 MHz) made by JapanElectron Optics Laboratory Co., Ltd.) under the above-mentionedmeasurement condition. From the result of GPC, it was known that PEGchain had a molecular weight of 5530, and a molecular weightdistribution Mw/Mn of 1.03. The molecular weight of lactide chain (PLA)of acetal-PEG/PLA-methacryloyl was found to be 150, as calculated fromthe molecular weight of PEG chain and ¹H-NMR spectrum which were theresults of GPC. From the ¹H-NMR spectrum, vinyl group-introducing ratewas calculated, and, thus, it was confirmed that vinyl group had beenalmost quantitatively introduced.

EXAMPLE 1 Preparation of Fluorescent Substance-EncapsuratedAmino-Terminated Core-Shell Type Latex

A vessel was fed with 0.4577 g (0.5 mmol) of europium (III) thenoyltrifluoroacetone (Eu-TTA), 0.3945 g (1 mmol) of trioctylphosphine oxide(TOPO) and 20 mg (0.12 mmol) of azobisisobutyronitrile (AIBN) in argonatmosphere. Then, 20 mL of methanol was added, and the resultant mixturewas dissolved by ultrasonic irradiation. Furthermore, 0.5 mL (4.35 mmol)of styrene monomer was added. The resultant solution is hereinafterreferred to as chelate monomer solution. In argon atmosphere, a vesselwas fed with 0.25 g (0.0487 mmol) of VB-PEG-NH₂ as obtained in MacromerSynthesis Example 1 and 20 mL of argon-deaerated super high-puritywater. To the resultant mixture, the above-mentioned chelate monomersolution was added with stirring by three-one-motor (500 r.p.m.). Afterfurther stirring at room temperature for 30 minutes, polymerizationreaction was made to occur by stirring at 60° C. for 24 hours. Theresultant particle suspension was dialyzed, and purified by centrifugalseparation (6000 r.p.m, 30 minutes, 4° C.). Further purification wasconducted by ultracentrifugal separation (80000 r.p.m, 20 minutes, 4°C.), and, finally, the suspension was subjected to filter treatment with0.45 μm hydrophilic membrane filter to give core-shell type latexaqueous suspension wherein amino group was bonded to surface, andwherein fluorescent substance was encapsulated in core portion.

Table-1 shows the result of calculation of fluorescent substanceaddition rate on the basis of the weight of particles used for thereaction, and of relative ratio of the addition rate (based onComparative Example 1 as mentioned later). The weight of total monomerused for the reaction of Examples of this invention corresponds to theweight of particles in Comparative Examples, for calculation.

Thus obtained core-shell type latex was measured for average particlesize and particle size distribution with the above-mentioned DynamicLight Scattering (DLS) Photometer (DLS-7000) made by Otsuka ElectronicsCo., Ltd., and for intensity of fluorescence with SpectrofluorophtometerF-2500 made by Hitachi, Ltd. Table-2 shows the ratio of intensity offluorescence per gram of particle (based on Comparative Example 1 asmentioned later) as well as average particle size and particle sizedistribution.

EXAMPLE 2

A vessel was fed with 0.4 g (0.0779 mmol) of VB-PEG-NH₂ as obtained inMacromer Synthesis Example 1, 0.4577 g (0.5 mmol) of europium (III)thenoyl trifluoroacetone (Eu-TTA), 0.3945 g (1 mmol) oftrioctylphosphine oxide (TOPO) and 20 mg (0.12 mol) ofazobisisobutyronitrile (AIBN) in argon atmosphere. Then, 20 mL ofmethanol was added, and the resultant mixture was dissolved byultrasonic irradiation. Furthermore, 0.5 mL (4.35 mmol) of styrenemonomer was added. To the resultant mixture, 20 mL of argon-deaeratedsuper high-purity water was added with stirring by three-one-motor (500r.p.m.). After stirring at room temperature for 30 minutes,polymerization reaction was made to occur by stirring at 60° C. for 24hours. The resultant particle suspension was dialyzed, and purified bycentrifugal separation (6000 r.p.m, 30 minutes, 4° C.). Finally, thesuspension was subjected to filter treatment with 0.45 μm hydrophilicmembrane filter to give core-shell type latex aqueous suspension whereinamino group was bonded to surface, and wherein fluorescent substance wasencapsulated in core portion. Obtained data are shown in Table-1 andTable-2 below, as in Example 1.

EXAMPLE 3

A vessel was fed with 0.12 g (0.0234 mmol) of VB-PEG-NH₂ as obtained inMacromer Synthesis Example 1, 0.28 g (0.0893 mmol) of VB-PEG-OH asobtained in Macromer Synthesis Example 3, 0.4577 g (0.5 mmol) ofeuropium (III) thenoyl trifluoroacetone (Eu-TTA), 0.3945 g (1 mmol) oftrioctylphosphine oxide (TOPO) and 20 mg (0.12 mol) ofazobisisobutyronitrile (AIBN) in argon atmosphere. Then, 20 mL ofmethanol was added, and the resultant mixture was dissolved byultrasonic irradiation. Furthermore, 0.5 mL (4.35 mmol) of styrenemonomer was added. To the resultant mixture, 20 mL of argon-deaeratedsuper high-purity water was added with stirring by three-one-motor (500r.p.m.). After stirring at room temperature for 30 minutes,polymerization reaction was made to occur by stirring at 60° C. for 24hours. The resultant particle suspension was dialyzed, and purified bycentrifugal separation (6000 r.p.m, 30 minutes, 4° C.). Finally, thesuspension was subjected to filter treatment with 0.45 μm hydrophilicmembrane filter to give core-shell type latex aqueous suspension whereinamino group was bonded to surface, and wherein fluorescent substance wasencapsulated in core portion. Obtained data are shown in Table-1 andTable-2 below, as in Example 1.

EXAMPLE 4

A vessel was fed with 0.04 g (0.00779 mmol) of VB-PEG-NH₂ as obtained inMacromer Synthesis Example 1, 0.36 g (0.115 mmol) of VB—PEG-OH asobtained in Macromer Synthesis Example 3, 0.4577 g (0.5 mmol) ofeuropium (III) thenoyl trifluoroacetone (Eu-TTA), 0.3945 g (1 mmol) oftrioctylphosphine oxide (TOPO) and 20 mg (0.12 mol) ofazobisisobutyronitrile (AIBN) in argon atmosphere. Then, 20 mL ofmethanol was added, and the resultant mixture was dissolved byultrasonic irradiation. Furthermore, 0.5 mL (4.35 mmol) of styrenemonomer was added. To the resultant mixture, 20 mL of argon-deaeratedsuper high-purity water was added with stirring by three-one-motor (500r.p.m.). After stirring at room temperature for 30 minutes,polymerization reaction was made to occur by stirring at 60° C. for 24hours. The resultant particle suspension was dialyzed, and purified bycentrifugal separation (6000 r.p.m, 30 minutes, 4° C.). Finally, thesuspension was subjected to filter treatment with 0.45 μm hydrophilicmembrane filter to give core-shell type latex aqueous suspension whereinamino group was bonded to surface, and wherein fluorescent substance wasencapsulated in core portion. Obtained data are shown in Table-1 andTable-2 below, as in Example 1.

EXAMPLE 5

A vessel was fed with 0.012 g (0.00234 mmol) of VB-PEG-NH₂ as obtainedin Macromer Synthesis Example 1, 0.388 g (0.124 mmol) of VB-PEG-OH asobtained in Macromer Synthesis Example 3, 0.4577 g (0.5 mmol) ofeuropium (III) thenoyl trifluoroacetone (Eu-TTA), 0.3945 g (1 mmol) oftrioctylphosphine oxide (TOPO) and 20 mg (0.12 mol) ofazobisisobutyronitrile (AIBN) in argon atmosphere. Then, 20 mL ofmethanol was added, and the resultant mixture was dissolved byultrasonic irradiation. Furthermore, 0.5 mL (4.35 mmol) of styrenemonomer was added. To the resultant mixture, 20 mL of argon-deaeratedsuper high-purity water was added with stirring by three-one-motor (500r.p.m.). After stirring at room temperature for 30 minutes,polymerization reaction was made to occur by stirring at 60° C. for 24hours. The resultant particle suspension was dialyzed, and purified bycentrifugal separation (6000 r.p.m, 30 minutes, 4° C.). Finally, thesuspension was subjected to filter treatment with 0.45 μm hydrophilicmembrane filter to give core-shell type latex aqueous suspension whereinamino group was bonded to surface, and wherein fluorescent substance wasencapsulated in core portion. Obtained data are shown in Table-1 andTable-2 below, as in Example 1.

REFERENTIAL EXAMPLE 1 Preparation of Core-Shell Type LatexAldehyde-Terminated Core-Shell Type Latex

A vessel was fed with 29.6 mg (0.18 mmol) of azobisisobutyronitrile(AIBN) in argon atmosphere. Then, 2 mL (17 mmol) of styrene solution wasadded. The resultant solution is hereinafter referred to as styrenesolution. Another vessel was fed with 160 mL of high-purity water and3.436 g (0.625 mmol) of acetal-PEG/PLA-methacryloyl as obtained inMacromer Synthesis Example 4, and, then, the air in vessel was replacedwith argon. The resultant solution is hereinafter referred to as monomersolution. While this solution was stirred by three-one-motor (400r.p.m.), the above-mentioned styrene solution was added. The resultantmixture was stirred at room temperature for 30 minutes, and then at 60°C. for 18 hours, and, furthermore, at 80° C. for 6 hours, and, thus,polymerization reaction was made to occur. The resultant particlesuspension was filtrated by filter paper to give core-shell type latexaqueous suspension wherein acetal group was bonded to surface.

The core-shell type latex aqueous suspension was adjusted to pH 2.0 with1M hydrochloric acid, and was then stirred for two hours. Subsequently,the solution was adjusted to pH 5.0 with 1M aqueous solution of sodiumhydroxide. Then, acetal group as protective group was deblocked to givecore-shell type latex aqueous suspension whose surface was now aldehydegroup.

This aqueous suspension was dialyzed, and filtrated with filter paper,and was then desalted.

Thus obtained aldehyde-terminated core-shell type latex was measured foraverage particle size and particle size distribution with theabove-mentioned Dynamic Light Scattering (DLS) Photometer (DLS-7000)made by Otsuka Electronics Co., Ltd. Particle size was 65 nm, andparticle size distribution was 0.151.

REFERENTIAL EXAMPLE 2 Aldehyde-Terminated Core-Shell Type Latex

A vessel was fed with 388.2 mg (2.4 mmol) of azobisisobutyronitrile(AIBN) in argon atmosphere. Then, 27 mL (235 mmol) of styrene solutionwas added. The resultant solution is hereinafter referred to as styrenesolution. Another vessel was fed with 400 mL of high-purity water and8.59 g (1.56 mmol) of acetal-PEG/PLA-methacryloyl as obtained inMacromer Synthesis Example 4, and, then, the air in vessel was replacedwith argon. The resultant solution is hereinafter referred to asmacromer solution. While this solution was stirred by three-one-motor(400 r.p.m.), the above-mentioned styrene monomer solution was added.The resultant mixture was stirred at room temperature for 30 minutes,and then at 60° C. for 18 hours, and, furthermore, at 80° C. for 6hours, and, thus, polymerization reaction was made to occur. Theresultant particle suspension was filtrated by filter paper to givecore-shell type latex aqueous suspension wherein acetal group was bondedto surface.

The core-shell type latex aqueous suspension was adjusted to pH 2.0 with1M hydrochloric acid, and was then stirred for two hours. Subsequently,the solution was adjusted to pH 5.0 with 1M aqueous solution of sodiumhydroxide. Then, acetal group as protective group was deblocked to givecore-shell type latex aqueous suspension whose surface was now aldehydegroup.

This aqueous suspension was dialyzed, and filtrated with filter paper,and was then desalted.

Thus obtained aldehyde-terminated core-shell type latex was measured foraverage particle size and particle size distribution with theabove-mentioned Dynamic Light Scattering (DLS) Photometer (DLS-7000)made by Otsuka Electronics Co., Ltd. Particle size was 102.3 nm, andparticle size distribution was 0.0665.

REFERENTIAL EXAMPLE 3 Amino-Terminated Core-Shell Type Latex

A vessel was fed with 20 mg (0.12 mmol) of azobisisobutyronitrile (AIBN)in argon atmosphere. Then, 0.5 mL (4.35 mmol) of styrene solution wasadded. The resultant solution is hereinafter referred to as styrenemonomer solution. Another vessel was fed with 0.25 g (0.0487 mmol) ofVB-PEG-NH₂ as obtained in Macromer Synthesis Example 1 in argonatmosphere, and then with 20 mL of argon-deaerated super high-puritywater. While this solution was stirred by three-one-motor (500 r.p.m.),the above-mentioned styrene monomer solution was added. The resultantmixture was stirred at room temperature for 30 minutes, and then at 60°C. for 20 hours (400 r.p.m.), and, furthermore, at 80° C. for fourhours, and, thus, polymerization reaction was made to occur. Theresultant particle aqueous suspension was filtrated by filter paper togive core-shell type latex aqueous suspension wherein amino group wasbonded to surface.

Thus obtained aldehyde-terminated core-shell type latex was measured foraverage particle size and particle size distribution with theabove-mentioned Dynamic Light Scattering (DLS) Photometer (DLS-7000)made by Otsuka Electronics Co., Ltd. Particle size was 98.2 nm, andparticle size distribution was 0.087.

COMPARATIVE EXAMPLE Preparation of Fluorescent Substance-EncapsuratedCore-Shell Type Latex by Swelling Action in Organic Solvent ComparativeExample 1

To 1 mL (22 mg/mL, 0.06 mmol) of europium chloride hexahydrate, therewas added 1 mL (37 mg/mL, 0.17 mmol) of acetone solution ofthenoyltrifluoroacetone (TTA), and, subsequently, 2 mL (87 mg/mL, 0.23mmol) of acetone solution of trioctylphosphine oxide (TOPO) was added,and, thus, europium chelate solution was prepared.

To 5 ml (18.13 mg/mL) of suspension of aldehyde-terminated core-shelltype latex of Referential Example 1, there was added 5 mL of acetone.Furthermore, with stirring, 0.12 mL of the above-mentioned europiumchelate solution (0.0018 mmol as europium chelate) was added, and theresultant mixture was stirred at room temperature under light shieldingfor 25 minutes. After stirring was over, acetone was evaporated withevaporator. Then, excessive europium chelate was removed by treatmentwith 0.2 μm hydrophilic membrane filter, and, thus, aldehyde-terminatedfluorescent substance-encapsulated core-shell type latex was obtained.

Table-1 shows the result of calculation of fluorescent substanceaddition rate on the basis of the weight of particles used for thereaction, and of relative ratio of the addition rate (based on thisExample). In this Comparative Example, the weight of particles used forreaction corresponds to the weight of total monomer used for reaction inExamples, for calculation.

Thus obtained core-shell type latex was measured for average particlesize and particle size distribution with the above-mentioned DynamicLight Scattering (DLS) Photometer (DLS-7000) made by Otsuka ElectronicsCo., Ltd., and for intensity of fluorescence with SpectrofluorophtometerF-2500 made by Hitachi, Ltd. Table-2 shows the ratio of intensity offluorescence per gram of particle (based on this Example).

Comparative Example 2

To 549.2 mg (0.60 mmol) of europium (III) thenoyl trifluoroacetone(Eu-TTA) and 473.4 mg (1.2 mmol) of trioctylphosphine oxide (TOPO),there was added 4 mL of acetone, and, thus, europium chelate solutionwas prepared.

To 10 mL (10.0 mg/mL, distilled water) of suspension ofaldehyde-terminated core-shell type latex of Referential Example 2,there was added 10 mL of acetone. Furthermore, with stirring, 0.24 mL ofthe above-mentioned europium chelate solution (0.036 mmol as europiumchelate) was added, and the resultant mixture was stirred at roomtemperature under light shielding for 30 minutes. After stirring wasover, acetone was evaporated with evaporator, and, then, the amount ofthe mixture was adjusted to 10 mL with super high-purity water by ameasuring pipet. The resultant aqueous solution was subjected tocentrifugal separation (3000 r.p.m, 30 minutes, 4° C.). Then, excessiveeuropium chelate was removed by treatment with 0.2 μm hydrophilicmembrane filter, and, thus, aldehyde-terminated fluorescentsubstance-encapsulated core-shell type latex was obtained. Data areshown in Table-1 and Table-2 below as in Example 1.

Comparative Example 3

To 32.96 mg (0.036 mmol) of europium (III) thenoyl trifluoroacetone(Eu-TTA) and 28.4 mg (0.072 mmol) of trioctylphosphine oxide (TOPO),there was added 10 mL of acetone, and, thus, europium chelate solutionwas prepared.

To this solution, 10 mL (10.0 mg/mL) of suspension ofaldehyde-terminated core-shell type latex of Referential Example 2 wasadded with stirring. The resultant mixture was stirred at roomtemperature under light shielding for 30 minutes. After stirring wasover, acetone was evaporated with evaporator, and, then, the amount ofthe mixture was adjusted to 10 mL with distilled water by a measuringpipet. The resultant aqueous solution was subjected to centrifugalseparation (3000 r.p.m, 30 minutes, 4° C.). Then, excessive europiumchelate was removed by treatment with 0.2 μm hydrophilic membranefilter, and, thus, aldehyde-terminated fluorescentsubstance-encapsulated core-shell type latex was obtained. Data areshown in Table-1 and Table-2 below as in Example 1.

Comparative Example 4

To 329.5 mg (0.36 mmol) of europium (III) thenoyl trifluoroacetone(Eu-TTA) and 284.1 mg (0.72 mmol) of trioctylphosphine oxide (TOPO),there was added 10 mL of acetone, and, thus, europium chelate solutionwas prepared.

To this solution, 10 mL (10.0 mg/mL) of suspension ofaldehyde-terminated core-shell type latex of Referential Example 2 wasadded with stirring. The resultant mixture was stirred at roomtemperature under light shielding for 30 minutes. After stirring wasover, acetone was evaporated with evaporator, and, then, the amount ofthe mixture was adjusted to 10 mL with distilled water by a measuringpipet. The resultant aqueous solution was subjected to centrifugalseparation (6000 r.p.m, 30 minutes, 4° C.). Then, excessive europiumchelate was removed by treatment with 0.2 μm hydrophilic membranefilter, and, thus, aldehyde-terminated fluorescentsubstance-encapsulated core-shell type latex was obtained. Data areshown in Table-1 and Table-2 below as in Example 1.

Comparative Example 5

To 164.8 mg (0.18 mmol) of europium (III) thenoyl trifluoroacetone(Eu-TTA) and 142.1 mg (0.36 mmol) of trioctylphosphine oxide (TOPO),there was added 5 mL of acetone, and, thus, europium chelate solutionwas prepared.

To this solution, 5 mL (10.0 mg/mL) of suspension of amino-terminatedcore-shell type latex of Referential Example 3 was added with stirring.The resultant mixture was stirred at room temperature under lightshielding for 30 minutes. After stirring was over, acetone wasevaporated with evaporator, and, then, the amount of the mixture wasadjusted to 15 mL with distilled water by a measuring pipet. Theresultant aqueous solution was subjected to centrifugal separation (6000r.p.m, 30 minutes, 4° C.). Then, excessive europium chelate was removedby treatment with 0.45 μm hydrophilic membrane filter, and, thus,aldehyde-terminated fluorescent substance-encapsulated core-shell typelatex was obtained. Data are shown in Table-1 and Table-2 below as inExample 1.

EXAMPLE 6 Preparation of Fluorescent Substance-EncapsulatedAldehyde-Terminated Core-Shell Type Latex

A vessel was fed with 0.2288 g (0.25 mmol) of europium (III) thenoyltrifluoroacetone (Eu-TTA), 0.1933 g (0.49 mmol) of trioctylphosphineoxide (TOPO) and 49.3 mg (0.30 mmol) of azobisisobutyronitrile (AIBN) inargon atmosphere. Then, 10 mL of acetone and 1 mL (8.70 mmol) of styrenesolution were added. The resultant solution is hereinafter referred toas styrene solution. Another vessel was fed with 80 mL of superhigh-purity water and 1.72 g (0.31 mmol) of acetal-PEG/PLA-methacryloylas obtained in Macromer Synthesis Example 4, and, then, the air invessel was replaced with argon. The resultant solution is hereinafterreferred to as monomer solution. While this solution was stirred bythree-one-motor (400 r.p.m.), the above-mentioned styrene monomersolution was added. The resultant mixture was stirred at roomtemperature for 30 minutes, and then at 60° C. for 24 hours (400r.p.m.), and, thus, polymerization reaction was made to occur. Afterpolymerization was over, acetone was evaporated by evaporator. Theresultant particle suspension was purified by centrifugal separation(2500 r.p.m, 30 minutes, 4° C.). Finally, the suspension was subjectedto filter treatment with 0.2 μm hydrophilic membrane filter to giveacetal-terminated fluorescent substance-encapsulated core-shell typelatex.

Then, the core-shell type latex solution was adjusted to pH 2.0 with 1Mhydrochloric acid, and was then stirred for two hours. Subsequently, thesolution was adjusted to pH 5.0 with 1M aqueous solution of sodiumhydroxide. Then, acetal group as protective group was deblocked to givecore-shell type latex aqueous suspension whose surface was now aldehydegroup.

This aqueous suspension was dialyzed, and filtrated with filter paper,and was then desalted. Data are shown in Table-1 and Table-2 below as inExample 1.

EXAMPLE 7 Preparation of Fluorescent Substance-EncapsulatedAcetal-Terminated Core-Shell Type Latex

A vessel was fed with 0.0572 g (0.062 mmol) of europium (III) thenoyltrifluoroacetone (Eu-TTA), 0.0483 g (0.12 mmol) of trioctylphosphineoxide (TOPO) and 20 mg (0.12 mmol) of azobisisobutyronitrile (AIBN) inargon atmosphere. Then, 5 mL of acetone and 1.3 mL (11.3 mmol) ofstyrene solution were added. The resultant solution is hereinafterreferred to as styrene solution. Another vessel was fed with 20 mL ofsuper high-purity water and 0.43 g (0.078 mmol) ofacetal-PEG/PLA-methacryloyl as obtained in Macromer Synthesis Example 4,and, then, the air in vessel was replaced with argon. The resultantsolution is hereinafter referred to as monomer solution. While thissolution was stirred by three-one-motor (400 r.p.m.), theabove-mentioned styrene monomer solution was added. The resultantmixture was stirred at room temperature for 30 minutes, and then at 60°C. for 24 hours (400 r.p.m.), and, thus, polymerization reaction wasmade to occur. After polymerization was over, acetone was evaporated byevaporator. The resultant particle suspension was purified bycentrifugal separation (10000 r.p.m, 30 minutes, 4° C.). Finally, thesuspension was subjected to filter treatment with 0.2 μm hydrophilicmembrane filter to give acetal-terminated fluorescentsubstance-encapsulated core-shell type latex. Data are shown in Table-1and Table-2 below as in Example 1.

EXAMPLE 8

A vessel was fed with 0.5721 g (0.62 mmol) of europium (III) thenoyltrifluoroacetone (Eu-TTA), 0.4833 g (1.23 mmol) of trioctylphosphineoxide (TOPO) and 20 mg (0.12 mmol) of azobisisobutyronitrile (AIBN) inargon atmosphere. Then, 5 mL of acetone and 1.3 mL (11.3 mmol) ofstyrene solution were added. The resultant solution is hereinafterreferred to as styrene solution. Another vessel was fed with 20 mL ofsuper high-purity water and 0.43 g (0.078 mmol) ofacetal-PEG/PLA-methacryloyl as obtained in Macromer Synthesis Example 4,and, then, the air in vessel was replaced with argon. The resultantsolution is hereinafter referred to as monomer solution. While thissolution was stirred by three-one-motor (400 r.p.m.), theabove-mentioned styrene monomer solution was added. The resultantmixture was stirred at room temperature for 30 minutes, and then at 60°C. for 24 hours (400 r.p.m.), and, thus, polymerization reaction wasmade to occur. After polymerization was over, acetone was evaporated byevaporator. The resultant particle suspension was purified bycentrifugal separation (10000 r.p.m, 30 minutes, 4° C.). Finally, thesuspension was subjected to filter treatment with 0.2 μm hydrophilicmembrane filter to give acetal-terminated fluorescentsubstance-encapsulated core-shell type latex. Data are shown in Table-1and Table-2 below as in Example 1.

EXAMPLE 9

A vessel was fed with 1.1441 g (1.25 mmol) of europium (III) thenoyltrifluoroacetone (Eu-TTA), 0.9666 g (2.45 mmol) of trioctylphosphineoxide (TOPO) and 20 mg (0.12 mmol) of azobisisobutyronitrile (AIBN) inargon atmosphere. Then, 5 mL of acetone and 1.3 mL (11.3 mmol) ofstyrene solution were added. The resultant solution is hereinafterreferred to as styrene solution. Another vessel was fed with 20 mL ofsuper high-purity water and 0.43 g (0.078 mmol) ofacetal-PEG/PLA-methacryloyl as obtained in Macromer Synthesis Example 4,and, then, the air in vessel was replaced with argon. The resultantsolution is hereinafter referred to as monomer solution. While thissolution was stirred by three-one-motor (400 r.p.m.), theabove-mentioned styrene monomer solution was added. The resultantmixture was stirred at room temperature for 30 minutes, and then at 60°C. for 24 hours (400 r.p.m.), and, thus, polymerization reaction wasmade to occur. After polymerization was over, acetone was evaporated byevaporator. The resultant particle suspension was purified bycentrifugal separation (10000 r.p.m, 30 minutes, 4° C.). Finally, thesuspension was subjected to filter treatment with 0.2 μm hydrophilicmembrane filter to give acetal-terminated fluorescentsubstance-encapsulated core-shell type latex. Data are shown in Table-1and Table-2 below as in Example 1.

TABLE 1 Fluorescent substance addition rate, and relative ratio of theamount of fluorescent substance added Amount of How fluorescent Relativeratio of the to introduce Surface substance Weight of Weight Fluorescentsubstance Fluorescent substance amount of fluorescent functional addedmonomer of particles addition addition fluorescent No. substance group(nmol) added (g) *1 (g) *2 rate (mmol/g) *3 rate (mmol/g) *4 substanceadded *5 Ex. 1 Method of this Amino 0.5 0.703 0.711 36 invention groupEx. 2 Method of this Amino 0.5 0.853 0.586 30 invention group Ex. 3Method of this Amino 0.5 0.853 0.586 30 invention group Ex. 4 Method ofthis Amino 0.5 0.853 0.586 30 invention group Ex. 5 Method of this Amino0.5 0.853 0.586 30 invention group CEx. 2 Swelling action Aldehyde 0.0180.1 0.180 9 by organic group solvent CEx. 3 Swelling action Aldehyde0.036 0.1 0.360 18 by organic group solvent CEx. 4 Swelling actionAldehyde 0.36 0.1 3.600 181 by organic group solvent CEx. 5 Swellingaction Amino 0.18 0.05 3.600 181 by organic group solvent CEx. 1Swelling action Aldehyde 0.0018 0.09 0.020 1 by organic group solventEx. 6 Method of this Aldehyde 0.25 2.626 0.095 5 invention group Ex. 7Method of this Acetal 0.062 1.608 0.039 2 invention group Ex. 8 Methodof this Acetal 0.62 1.608 0.386 19 invention group Ex. 9 Method of thisAcetal 1.25 1.608 0.777 39 invention group Ex.: Example CEx.:Comparative Example *1: Total weight of particle-constituting maincomponent (macromer + styrene) added (corresponding to the weight ofparticles used for the reaction of Comparative Examples 1 to 5) *2:Weight of particles used for the reaction *3: Molar amount offluorescent substance added per gram of particle-constituting maincomponent (macromer + styrene) used for the reaction *4: Molar amount offluorescent substance added per gram of particles used for the reaction*5: Relative ratio of the amount of fluorescent substance added ascompared with Comparative Example 1

TABLE 2 Average particle size and the ratio of intensity of fluorescenceHow Relative ratio of the to introduce Surface amount of AverageParticle Excitation Wave length of fluorescent functional fluorescentparticle size wave fluorescence Ratio of intensity of No. substancegroup substance added *1 size (nm) distribution length (nm) (nm)fluorescence *2 Ex. 1 Method of this Amino 36 162.7 0.056 340 616 8.1invention group Ex. 2 Method of this Amino 30 163.2 0.037 340 616.5 8.3invention group Ex. 3 Method of this Amino 30 160.9 0.115 340 616 10.6invention group Ex. 4 Method of this Amino 30 147.3 0.094 340 616 9.6invention group Ex. 5 Method of this Amino 30 150.9 0.088 340 616.5 10.1invention group CEx. 2 Swelling Aldehyde 9 106.7 0.043 340 616.5 1.6action by group organic solvent CEx. 3 Swelling Aldehyde 18 107.9 0.048340 616.5 6.1 action by group organic solvent CEx. 4 Swelling Aldehyde181 123.2 0.089 340 616 7.8 action by group organic solvent CEx. 5Swelling Amino 181 107.9 0.039 340 615 7.1 action by group organicsolvent CEx. 1 Swelling Aldehyde 1 65.1 0.078 340 616.5 1.0 action bygroup organic solvent Ex. 6 Method of this Aldehyde 5 52.7 0.120 340616.5 0.8 invention group Ex. 7 Method of this Acetal 2 121.0 0.064 340616.5 1.9 invention group Ex. 8 Method of this Acetal 19 118.8 0.041 340616.5 0.6 invention group Ex. 9 Method of this Acetal 39 81.8 0.057 340616.5 1.6 invention group *1: Relative ratio of the amount offluorescent substance added as compared with Comparative Example 1 *2:Relative ratio of the intensity of fluorescence per gram of particle ascompared with Comparative Example 1

It is known from the above-mentioned data that the process of thisinvention to manufacture latex polymer particles provides latex polymerparticles which efficiently and stably contain fluorescent substance orcontrast medium.

EXAMPLE 10

Each of aqueous particle suspensions which had been obtained accordingto the methods of Examples 1 to 5 and Comparative Examples 1 to 5 wasadjusted to a particle concentration of 2 mg/mL with super high-puritywater. Another each of said aqueous particle suspensions was adjustedwith super high-purity water so that the intensity of fluorescence asmeasured might be about 15000. Then, each of thus prepared aqueousparticle suspensions which had been unified with regard to eitherparticle concentration or the intensity of fluorescence was diluted to1/500 with a buffer (1/15 M PBS, pH 7.4, which contained 0.05 wt %sodium dodecyl sulfate (SDS)), and was subsequently measured for theintensity of fluorescence by time-resolved fluorescence measurementunder the following measuring conditions. Later, the suspensions werestored at 37° C., and were measured for the intensity of fluorescenceunder the same conditions after three days, five days and seven days.The intensity of fluorescence was measured three times for eachmeasurement, and, so, an average was obtained from thus measured threevalues. With use of this average value, the rate of change of theintensity of fluorescence with lapse of time was calculated, based onthe intensity of fluorescence immediately after the dilution with bufferas a standard (100%), and, thus, the particles were compared withrespect to fluorescence stability.

<Measurement Apparatus and Conditions etc.>

-   Multi-detection Microplate Reader Power Scan HT made by Dainippon    Pharmaceutical Co., Ltd.-   Light Source: 10 W Xenon Flash Lamp, Excitation Wave Length: 340 nm,    Fluorescence Wave Length: Standard-   Interference Filter 620/40 nm, Sensitivity: 120-   Measurement Times: 50 times-   Time span between transfer to the location of measurement and    irradiation from light source: 500 μsec-   Measurement Interval: 10 msec-   Retardation Time: 200 μsec-   Measurement Time: 400 μsec    <Results>

Table-3 shows the intensity of fluorescence when particle concentrationwas adjusted to 2 mg/mL, and how the intensity of fluorescence changedwith lapse of time. Initial intensity of fluorescence was clearly higherin Examples 1 to 5 than in Comparative Examples 1 to 5. With regard tohow the intensity of fluorescence changed with lapse of time, 90% ormore of the initial intensity was maintained at 37° C. even after sevendays in Examples. In Comparative Examples, however, the intensitylowered to 62 to 85%. This result shows that the fluorescence inExamples was more stable.

Table-4 shows how the intensity of fluorescence changed with lapse oftime when the initial intensity of fluorescence was unified at about15000. Whereas, in Examples, 95% or more of the initial intensity wasmaintained at 37° C. after seven days, it lowered to 78 to 84% inComparative Examples. This result shows that the particles of Exampleswere more stable with lapse of time than those of Comparative Examples.

TABLE 3 Evaluation of fluorescence stability (unified particleconcentration: 2 mg/mL) Average of fluorescence intensity at After 3days After 5 days After 7 days Examples time 0 (%) (%) (%) Ex. 1 1662592.5 ± 1.9 90.5 ± 2.8 91.6 ± 1.8 Ex. 2 15003 95.0 ± 1.5 93.3 ± 3.3 94.6± 3.1 Ex. 3 17746 98.6 ± 1.2 96.2 ± 3.2 97.2 ± 2.1 Ex. 4 17381 97.9 ±2.2 96.5 ± 3.0 96.4 ± 0.7 Ex. 5 15873 98.6 ± 1.1 95.2 ± 3.7 98.2 ± 3.5CEx. 1 950 68.8 ± 2.2 63.7 ± 1.9 62.1 ± 2.8 CEx. 2 4382 86.5 ± 3.4 77.6± 3.9 81.1 ± 3.1 CEx. 3 9483 89.4 ± 3.9 76.8 ± 5.2 78.8 ± 3.0 CEx. 411869 91.0 ± 3.6 82.3 ± 4.3 84.5 ± 3.5 CEx. 5 14073 93.2 ± 2.2 85.6 ±0.9 84.2 ± 2.9

TABLE 4 Evaluation of fluorescence stability (unified fluorescenceintensity: about 15000 count) Average of fluorescence intensity at After3 days After 5 days After 7 days Examples time 0 (%) (%) (%) Ex. 1 1675697.2 ± 2.3 93.8 ± 1.7 95.5 ± 2.7 Ex. 2 13651 98.6 ± 1.4 95.3 ± 2.8 97.8± 2.6 Ex. 3 12896 102.4 ± 0.6  96.9 ± 3.8 101.0 ± 2.5  Ex. 4 14923 103.1± 1.2  98.8 ± 2.1 100.5 ± 2.5  Ex. 5 15317  100 ± 2.5 97.7 ± 2.3 96.7 ±3.1 CEx. 1 15314 79.6 ± 0.7 76.1 ± 1.1 78.2 ± 2.8 CEx. 2 14863 79.6 ±2.4 82.8 ± 2.4 81.2 ± 3.1 CEx. 3 12786 84.2 ± 2.3 83.4 ± 1.7 84.1 ± 2.7CEx. 4 13619 93.8 ± 2.3 77.7 ± 2.6 78.0 ± 3.2 CEx. 5 13608 83.9 ± 1.880.5 ± 2.1 81.8 ± 2.1

INDUSTRIAL APPLICABILITY

The process of this invention provides latex polymer particles whichefficiently and stably contain fluorescent substance or contrast medium.Hence, not restrictively, this invention is applicable in medical fieldand in the field of manufacture of diagnostic medicines.

1. A process for producing fluorescent substance—or inorganic contrastmedium—containing latex polymer particles, which comprises conducting apolymerization reaction in an aqueous medium while the aqueous medium isstirred, said aqueous medium comprising: (i) one or more latex-formingmonomers, (ii) a macromer which has, on one terminal, a polymerizableethylenic group and has, on the other terminal, a hydrophilic polymersegment which is linked or not linked by a hydrophobic polymer segment,(iii) a radical polymerization initiator, and (iv) an inorganicfluorescent substance or an inorganic contrast medium.
 2. A process ofclaim 1 wherein hydrophilic polymer segment is originated fromwater-soluble polymer which is selected from the group consisting ofpoly(ethyleneglycol), poly(vinylalcohol), poly(vinylpyrrolidone),poly(dextran), poly(dextrin) and gelatin, and wherein hydrophobicpolymer segment is originated from scarcely water-soluble polymerselected from the group consisting of poly(lactide),poly(ε-caprolactone), poly(α- and/or β-benzyl aspartic acid) andpoly(γ-benzyl glutamic acid).
 3. A process of claim 1 wherein macromerhas no hydrophobic polymer segment, and wherein hydrophilic polymersegment is originated from poly(ethyleneglycol).
 4. A process of claim 1wherein macromer is of two or more kinds each of which has nohydrophobic polymer segment, and each of which has, at the otherterminal, a poly(ethyleneglycol) segment which carries a group selectedfrom the group consisting of hydroxyl group, carboxyl group, aldehydegroup, amino group, imino group, mercapto group, active ester-typeprotected hydroxyl group, active ester-type protected carboxyl group,acetal-type protected aldehyde group, organic sulfonyl-protectedhydroxyl group, reactivity-protected amino group and C₁-C₄ alkoxylgroup.
 5. A process of claim 1 wherein there are two macromers, thefirst macromer having no hydrophobic polymer segment, and having, at theother terminal, a poly(ethyleneglycol) segment which carries a groupselected from the group consisting of hydroxyl group and C₁-C₄ alkoxylgroup, the second macromer having no hydrophobic polymer segment, andhaving, at the other terminal, a poly(ethyleneglycol) segment whichcarries a group selected from the group consisting of carboxyl group,aldehyde group, amino group, imino group, mercapto group, activeester-type protected hydroxyl group, active ester-type protectedcarboxyl group, acetal-type protected aldehyde group,reactivity-protected amino group and organic sulfonyl-protected hydroxylgroup, said segment of the first macromer having a chain length which isthe same as, or shorter than, the chain length of said segment of thesecond macromer, and the molar proportion of the first macromer to thesecond macromer ranging from 1:5000 to 5000:1.
 6. A process of claim 5wherein the number of recurring unit of ethyleneglycol in the firstmacromer is an integer of 5 to 1200, the number of recurring unit ofethyleneglycol in the second macromer is an integer of 5 to 1200, andwherein the number of the first macromer recurring unit is the same as,or smaller than, the number of the second macromer recurring unit.
 7. Aprocess of any one of claims 1 to 5 wherein one or more kinds oflatex-forming monomers are selected from the group consisting ofstyrene, α-methylstyrene, p-bromostyrene, vinyltoluene,1-vinylnaphthalene, C₁-C₄ alkyl (meth)acrylate and divinyl benzene.
 8. Aprocess of claim 1 wherein inorganic fluorescent substance and inorganiccontrast medium are in the form of chelate compound.
 9. A process ofclaim 1 wherein macromer has general formula (I) as follows:

wherein R denotes hydrogen atom or C₁-C₄ alkyl group; L₁ denotes alinking group comprising a portion other than vinyl group of radicallypolymerizable monomer; B denotes a moiety of a structure selected fromthe following:

L₂ denotes a linking group comprising oxygen atom, C₁-C₄ alkylene,carbonyl, imino, or a combination of at least two thereof; X denoteshydrogen atom, C₁-C₄ alkyl, C₁-C₄ alkylenecarboxyl, C₁-C₄alkylenecarboxyl ester, C₁-C₄ alkylenecarboxylic acid halide, C₁-C₄alkyleneamino, C₁-C₄ alkylenemercapto, C₁-C₄ alkyleneacetal, C₁-C₄alkyleneoxycarbonylimidazol; m denotes an integer of 0 to 500, and ndenotes an integer of 5 to 1200.